Oxidative stress has been found to be associated with various disease states. An analytical tool which can be used to monitor cellular and subcellular oxidative stress in intact cells will be a valuable tool in understanding how cells respond to oxidative stress stimuli and help develop treatments to oxidative stress- associated diseases. A combination of a proper fluorogenic or fluorescent agent with laser scanning confocal microscopy (LSCM) has been widely used to image the distribution of the molecule of interest in different subcellular structures in intact cells and will be a method of choice in monitoring oxidative stress at different subcellular locations. Thiol (-SH) status plays a significant role in determining cellular oxidative stress status due to its high susceptibility to oxidation and high abundance. Thiol density decreases as oxidative stress increases and its status has been used as a parameter to reflect cellular oxidative stress. A proper thiol specific fluorogenic or fluorescent agent in combination with LSCM can be used to image thiol density and distribution in intact cells, which in turns reflects redox state at subcellular levels. Numerous fluorogenic and fluorescent agents have been developed for thiol assays. However, most of these agents are not thiol specific. Their use for thiol assays needs to be coupled with a separation technique, such as high performance liquid chromatography (HPLC) or liquid chromatography/mass spectrometry (LC/MS), and is not suitable for LSCM. Consequently, their use is limited to thiol assays of tissue homogenates. Only a few fluorogenic agents are available for thiol assays in intact cells by LSCM with various drawbacks. In view of the significant role oxidative stress plays in various cellular functions, disease states, and valuable information which can be gained from an intact cell study of thiol distribution, it is highly desirable to develop thiol specific fluorogenic agents which can be used for LSCM. The aim of this proposed application is to develop thiol specific fluorogenic agents which can be used to image thiol density intracellularly and on the cell surface in intact cells with LSCM. The design of these agents is based on a thiol specific reaction: thiol-disulfide (-S-S-) exchange reaction. This reaction is the base of the most widely used thiol specific reagent, Ellman's reagent which enables measurement of thiols through a visible wavelength. This proposed project is to take advantage of the thiol specific reaction and integrate the disulfide bond (-S-S-) into a well studied and widely used benzofurazan fluorogenic structure. Specifically, rationally designed thiol specific fluorogenic agents and their thiol adducts will be synthesized. Their fluorescence properties will be fully characterized. Their application in determining intracellular thiol distribution and thiol density on the cell surface in intact cells will be validated by LSCM with Chinese hamster ovary (CHO) cells under different extents of thiol oxidative stress. PUBLIC HEALTH RELEVANCE: Oxidative stress has been associated with various health issues. A method which can detect the status of oxidative stress in intact cells will help understand the role of oxidative stress in various diseases and facilitate development of treatments to oxidative stress-associated diseases. This proposed application is aimed to develop a method which can be used to detect the status of oxidative stress in intact cells.